This competitive renewal proposes to continue our work on the post-transcriptional regulation of the hundreds of human mRNAs that are controlled by the double-stranded (ds)RNA-binding protein Staufen (STAU). Over the last <4 years we have followed up our discovery of STAU1-mediated mRNA decay (SMD). During SMD, mRNAs that harbor a STAU1-binding site (SBS) in their 3'-untranslated regions (3'UTRs) are degraded when translation terminates (usually normally, i.e., not prematurely) upstream of the SBS. We have shown that SMD is critical for cell motility, myogenesis and probably most cellular processes. We have expanded what defines a human SBS to include intermolecular duplexes formed by base-pairing between an Alu element within a mRNA 3'UTR and a complementary Alu element within (i) one or more cytoplasmic long non-coding (lnc)RNAs, thereby defining new roles for Alu elements and lncRNAs, which we call 1/2sbs-RNAs, and/or (ii) one or more mRNAs, thereby defining an exciting new function for mRNAs. Alu elements, which are a type of small interspersed element (SINE), are restricted to primates. Our finding that these new and complex Alu functions also apply to the SINEs of mouse, coupled with our computational and statistical analyses, support the notion that orthologous human and mouse genes have independently acquired and maintained 3'UTR SINEs for the purpose of triggering SMD. In related studies, we have determined the X-ray crystal structure of the human STAU1 dimerization interface, which involves interactions between a new motif that we call the STAU- swapping motif (SSM) and a degenerate dsRNA-binding domain. We have reported that STAU1 dimerization promotes SMD by augmenting STAU binding to the RNA helicase UPF1. In fact, both STAU1 and its paralog STAU2 activate SMD by activating UPF1 helicase activity in a mechanism that requires ATP hydrolysis without increasing UPF1 ATPase activity. We have found that STAU1 also binds inverted-repeated Alu elements (IRAlus) within mRNA 3'UTRs to allow mRNA export to, and translation in, the cytoplasm rather than activate SMD. In this proposal, AIM 1 is to identify new human-gene products that mediate SMD using a haploid genetic approach. This approach is an important complementation to our on-going identification of proteins (using mass spectrometry) and, when appropriate, 1/2sbs-RNAs and mRNAs (using RNA-seq) that associate with the SBSs of individual SMD targets. AIM 2 is to define how human Staufen proteins affect RNA metabolism via (i) 3'UTR IRAlus, and (ii) the convergent evolution of human and mouse SINEs. AIM 3 will mine RNA-seq data identifying mRNAs that (i) are upregulated by 1/2sbs-RNA1 siRNA or 1/2sbs-RNA2 siRNA - data indicating that 1/2sbs-RNA1 regulates Wnt signaling - and/or (ii) co-purify with either 1/2sbs-RNA. This aim will also examine the mechanism of STAU-mediated UPF1 activation. In sum, we have shown that STAU is key to several post-transcriptional regulatory pathways affecting hundreds of human genes that, when improperly expressed, cause disease. We will continue to unravel how STAU paralogs contribute to cellular homeostasis.